WO2024079325A1 - Composition d'administration orale obtenue à partir de lysats de micro-organismes probiotiques destinés à être utilisés en tant qu'agent anti-vieillissement - Google Patents

Composition d'administration orale obtenue à partir de lysats de micro-organismes probiotiques destinés à être utilisés en tant qu'agent anti-vieillissement Download PDF

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WO2024079325A1
WO2024079325A1 PCT/EP2023/078516 EP2023078516W WO2024079325A1 WO 2024079325 A1 WO2024079325 A1 WO 2024079325A1 EP 2023078516 W EP2023078516 W EP 2023078516W WO 2024079325 A1 WO2024079325 A1 WO 2024079325A1
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composition
lactobacillus
genus
bacterial lysates
lysates
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PCT/EP2023/078516
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English (en)
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María Jesús GUARDIA ALBA
Juan MOSCOSO DEL PRADO UCELAY
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Igen Biolab Group Ag
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Publication of WO2024079325A1 publication Critical patent/WO2024079325A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/32—Manganese; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast

Definitions

  • the present invention describes a composition comprising lysates of probiotic microorganisms in the form of dry powder as an anti-aging agent and is highly safe even with a long period of consumption.
  • a composition is used as a food supplement for the prevention and delay of aging in humans.
  • the present invention is encompassed in the technical field of therapies whose object it is to delay or prevent the development of decreased energy, change of appearance in humans and animals due to aging.
  • Aging is a universal multifactorial and progressive intrinsic process characterized by being degenerative and accompanied by a progressive loss of function.
  • the functional capacity of a person begins to decline after he has reached adulthood, then producing aging that can be defined as a biological process by which living beings undergo a series of structural and functional changes that appear over time and are not the result of diseases or accidents.
  • joint problems can appear, affecting flexibility, loss of muscle mass causing fatigue, and reduction of bone mineral density, increasing the likelihood of fractures.
  • the rate of decline in a person's functional capacity appears to be determined, at least in part, by their behavior and the environmental living conditions to which he or she is exposed throughout their life.
  • Influential factors are diet, physical activity, and exposure to risks such as smoking, the harmful consumption of alcohol, or exposure to toxic substances.
  • One of the most frequent recommendations to counteract, mitigate and/or slow down the effects of aging as much as possible and improve healthy life expectancy is that people perform adequate physical activity, and follow a healthy diet based on vegetables, fruits, whole grains, foods with a lot of fiber, and sources of protein without fat, such as fish, with natural antioxidants and low saturated fat and salt.
  • Another theory explaining the aging process is the accumulation of reactive oxygen species that damage proteins, lipids and DNA by accumulating in cells.
  • the efficiency of endogenous antioxidant systems decreases, the presence of antioxidants is not enough to compensate reactive oxygen species, which generates oxidative stress that plays an important role in aging and age-related diseases such as certain types of cancer, diabetes and heart failure, and in the pathogenesis of gastrointestinal diseases, among others.
  • Numerous studies have shown that resistance to oxidative stress is crucial for staying healthy and reducing the adverse effects of aging. Consequently, nutritional interventions using food-grade antioxidant compounds or food products are considered as an option to help improve health and quality of life in the elderly.
  • there is currently no type of therapy that has been shown to be really effective in delaying the aging process.
  • Patent number US8492353B2 provides an anti-aging composition containing at least one selected member of the group consisting of ascorbic acid, a derivative of ascorbic acid and a salt thereof (A), and a substance related to purine nucleic acid (B) and which is capable of effectively retarding skin aging, in particular relieving skin pigmentation.
  • the present invention also provides a method to enhance the anti-aging action of ascorbic acids.
  • the invention provides an anti-aging composition.
  • Patent application US2020345694A1 concerns an anti-aging composition containing cytochalasin D or SAG, and a method of selecting an anti-aging substance, and provides a new anti-aging substance and a new method of discovering a new anti-aging substance.
  • Patent application JP2003155234A describes an anti-aging composition
  • a methyl group donor compound particularly betaine
  • the composition suppresses the increase of homocysteine in the blood and prevents the aging of the human body caused by the increase in homocysteine, both in oral and percutaneous administration.
  • the present invention aims to solve the problems of the prior art by using a composition of oral administration obtained from lysates of probiotic microorganisms, for use in the prevention and delay of aging in humans.
  • the present invention relates to the composition of oral administration to prevent and delay aging, that comprises lysates of probiotic microorganisms in the form of dry powder, in the following amount in percentage by weight with respect to the total:
  • the invention relates to the use of the composition object of the invention as a food supplement for the prevention and delay of aging in animal or human cells.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective pharmaceutical amount of the composition, in accordance with the first aspect of the invention, and a pharmaceutically acceptable excipient.
  • the invention relates to the use of a composition of oral administration to prevent and delay aging, that comprises lysates of probiotic microorganisms in the form of dry powder, in the following amount in percentage by weight with respect to the total:
  • FIG. 1 Standard curve of the protein Ct values of HeLa cells versus the logarithm of the protein (ng). The cycle number at the threshold (Ct value) for each sample is interpolated onto the curve to calculate the relative activity of telomerase (RTA).
  • FIG. 1 Q-TRAP results bar chart (RTA) for in vitro treatments in primary fibroblast cultures. Data were pooled by condition and time of treatment.
  • Figure 3. Growth curves of untreated adult primary human fibroblasts (control- DMSO) and treated with IG (IG_1 , IG_2, IG_3) under standard conditions. Each point on the population curve represents the average of the triplicates for each cell passage.
  • FIG. 1 Growth curves of untreated adult primary human fibroblasts (control-MSO) and treated IG (IG_1 , IG_2, IG_3) under oxidative conditions (H2O2 10 pM). Each point on the population curve represents the average triplicate for each cell step.
  • the histogram shows the distribution of telomere lengths in a representative sample.
  • the bars represent the relative frequency for each particular normalized fluorescence intensity (X-axis).
  • the 20th percentile (red bars) indicates the particular length below which 20% of telomeres have been observed.
  • the median (MTL) and mean length (ATL) of telomeres are also indicated in the histogram. This histogram also allows the analysis of telomere length variability.
  • Fig. 7. a Median telomere length.
  • Fig. 7.b 20th percentile.
  • Fig. 7.c % of telomeres of ⁇ 3kbp.
  • Fig. 8. a Median telomere length.
  • Fig. 8.b 20th percentile.
  • Fig. 8.c % of telomeres of ⁇ 3kbp, for the different treatments.
  • Figure 9 Bar graphs for the telomere shortening ratio for different times and treatments under standard conditions.
  • Figure 10 Bar graphs of shortening of the telomere ratio for the different treatments and times under conditions of oxidative stress.
  • Fig. 11 is a graph showing the effect of the composition in accordance with the invention at low doses on the longevity of C. elegans (N2).
  • Figure 12 is a graph showing the effect of the composition in accordance with the invention in intermediate doses on the longevity of C. elegans (N2).
  • Figure 13 is a graph showing the effect of the composition in accordance with the invention at high doses on the longevity of C. elegans (N2).
  • Figure 14 shows survival curves of C. elegans (N2) obtained in populations treated with the composition in accordance with the invention (1 mg/mL) by the computer program GraphPad Prism.
  • Figure 15 shows the average mobility of populations of C. elegans treated with the composition in accordance with the invention at different doses.
  • Figure 16 shows the average mobility of populations of C. elegans treated with the composition in accordance with the invention at doses 0.1 ; 0.5 and 1 mg/mL.
  • Figure 17 shows the frequency of curvatures of populations of C. elegans treated with the composition in accordance with the invention at doses 0.1 ; 0.5 and 1 mg/mL.
  • Figure 18 shows the degree of dispersion of worms C. elegans treated with the composition in accordance with the invention at doses 0.1 ; 0.5 and 1 mg/mL.
  • Figure 19 shows the antioxidant activity of the composition according to the invention evaluated at different doses (10-400 pg/mL) in C. elegans N2. The percentages of survival after applying oxidative stress with H2O2 are shown. Vitamin C (10 pg/mL) was included as a positive control. The data are for a single trial.
  • Figure 20 shows the antioxidant activity of the composition in accordance with the invention evaluated at different doses in C. elegans N2. The percentages of survival after applying oxidative stress with H2O2 are shown. Vitamin C (10 pg/mL) was included as a positive control. Data are for the mean of 2 independent trials.
  • Figure 21 represents the relative percentage increase in survival of C. elegans with the effective dose of the composition in accordance with the invention versus control condition, indicating an increase of 88.5%.
  • the inventors have detected the need to develop a composition that is administered as an anti-aging agent, that is easy to administer, and that does not present toxicity.
  • compositions that is administered orally, comprising lysates of microorganisms that is accompanied by other components for this composition, and that has turned out to be strikingly beneficial for humans and animals as it presents an antioxidant character and proves to be a promoter of cell proliferation, which makes it a highly effective anti-aging agent.
  • the present invention relates to a composition of oral administration to prevent and delay aging, that it comprises lysates of probiotic microorganisms in the form of dry powder, in the following amount in percentage by weight with respect to the total:
  • the composition can comprise between 20% - 99.5% by weight of bacterial lysates, particularly 25%-95% by weight of bacterial lysates, and more particularly 50% + 10% by weight of bacterial lysates.
  • bacterial lysates are understood as the product obtained after the process of cultivating, and subsequently, mechanically or chemically breaking said bacterial cells in order to obtain a product with microbial fragments, as well as all the components comprised therein.
  • these are dry bacterial lysates, since, thanks to their method of obtainment, these lysates are subsequently subjected to drying techniques, in such a way that said dry bacterial lysates are in powder form.
  • the bacterial lysates of the genus Bacillus are of the species that are selected from the group consisting of Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus mesentericus, Bacillus subtilis, and combinations thereof, or at least, Bacillus coagulans, Bacillus licheniformis, Bacillus mesentericus, Bacillus subtilis, and, optionally Bacillus clausii
  • the bacterial lysates of the genus Bifidobacterium are of the species that are selected from the group consisting of Bifidobacterium animalis subsp lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacter ium animalis, Bacillus paralicheniformis, and combinations thereof, or at least Bifidobacterium breve, and Bifidobacterium lactis and, optionally, Bifidobacterium bifidum.
  • the bacterial lysates of the genus Lactobacillus are of the species that are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus kefiri, and combinations thereof or at least Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and, optionally, Lactobacillus fermentum.
  • the bacterial lysates of the genus Saccharomyces are selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, and combinations thereof.
  • bacterial lysates of the genus Streptococcus are of the species Streptococcus thermophilus, Streptococcus salivarius, and combinations thereof.
  • the composition object of the invention comprises bacterial lysates of the genus Bacillus in an amount in percentage by weight between 5% to 30%, preferably between 8% to 25%, and even more preferably between 10% to 18%.
  • the amount of bacterial lysates of the genus Bacillus can be 5%, 6%, 7%, 8%, 9% or 10%.
  • the amount of bacterial lysates of the genus Bacillus can be 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%.
  • the composition object of the invention comprises bacterial lysates of the genus Bifidobacterium in an amount in percentage by weight between 3% to 25%, preferably between 5% to 20%, and even more preferably between 8% to 15%.
  • the amount of bacterial lysates of the genus Bifidobacterium can be 3%, 4% 5%, 6%, 7%, 8%, 9% or 10%.
  • the amount of bacterial lysates of the genus Bifidobacterium can be 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%.
  • the composition object of the invention comprises bacterial lysates of the genus Lactobacillus in an amount in percentage by weight between 15% to 35%, preferably between 15% to 30%, and even more preferably between 20% to 25%.
  • the amount of bacterial lysates of the genus Lactobacillus can be 15%, 16%, 17%, 18%, 19% or 20%.
  • the amount of bacterial lysates of the genus Lactobacillus can be 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35%.
  • the composition comprises bacterial lysates of the genus Saccharomyces in an amount in percentage by weight between 30% to 65%, preferably between 50% - 58%, and even more preferably between 35% - 60%.
  • the amount of bacterial lysates of the genus Saccharomyces can be 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% or 49%.
  • the amount of bacterial lysates of the genus Saccharomyces can be 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%.
  • the composition comprises bacterial lysates of the genus Streptococcus in an amount in percentage by weight between 1.5% to 10%, preferably between 2% to 7%, and even more preferably between 3% to 6%.
  • the amount of bacterial lysates of the genus Streptococcus can be 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3%.
  • the amount of bacterial lysates of the genus Streptococcus can be 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • composition A - approximately 2% to 8% of bacterial lysates of the genus Streptococcus. This composition is called composition "A'
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • composition "B” This composition is called composition "B"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • composition "C” This composition is called composition "C"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • Lactobacillus fermentunr - 15% to 35% of bacterial lysates of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and, optionally, Lactobacillus fermentunr,
  • composition "D" This composition is called composition "D"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • Lactobacillus fermentunr - 17% to 30% of bacterial lysates of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and, optionally, Lactobacillus fermentunr,
  • composition "E” This composition is called composition "E"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • Lactobacillus acidophilus Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus rhamnosus, and, optionally, Lactobacillus fermentunr,
  • composition "F" This composition is called composition "F"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • Bacillus coagulans 0.1% to 10%; Bacillus licheniformis 1% to 12%; Bacillus mesentericusVVo to 12%; Bacillus subtilis 0.1% to 10%; and, optionally Bacillus clausii 0.1 % to 5%;
  • Lactobacillus acidophilus 0.3% to 15%; Lactobacillus bulgaricus 0.3% to 15%; Lactobacillus casei 0.3% to 12%; Lactobacillus reuteri 0.5% to 10%; and Lactobacillus rhamnosus 0.1% to 10%; and, optionally, Lactobacillus fermentum 0.5% to 10%;
  • composition "G" This composition is called composition "G"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition:
  • Bacillus coagulans 0.1% to 6%; Bacillus licheniformis 2% to 10%; Bacillus mesentericus 2% to 10%; Bacillus subtilis 0.3% to 8%; and, optionally Bacillus clausii 0.1% to 4%;
  • composition "H” This composition is called composition "H"
  • the composition for use comprises lysates of probiotic microorganisms in an amount in percentage by weight of the total lysates of the composition: - Bacillus coagulans 0.1 % to 4%; Bacillus licheniformis 3% to 8%; Bacillus mesentericus 3% to 8%; Bacillus subtilis 0.1% to 10%; and, optionally Bacillus clausii 0.5% to 7%;
  • Lactobacillus acidophilus 2% to 10%; Lactobacillus bulgaricus 2% to 10%; Lactobacillus casei 2% to 8%; Lactobacillus reuteri 1.5% to 5%; and Lactobacillus rhamnosus 0.5% to 5%; and, optionally, Lactobacillus fermentum 2% to 5%;
  • composition "I” This composition is called composition "I"
  • the composition for use comprises the lysates of probiotic microorganisms in the form of dry powder, in the amount as a percentage by weight of the total as disclosed in compositions “A”, “B”, “C”, “D” E”, “F”, , ri or i .
  • composition object of the invention may comprise another additional component or additive that is selected from the group consisting of Riboflavin, Vitamin C, Manganese, Selenium, Zinc, Vitamin D and Magnesium.
  • Vitamin C contributes to the protection of cells against oxidative damage.
  • Vitamin D, Manganese and Zinc contribute to the maintenance of normal bones.
  • Vitamin D and Magnesium contribute to the normal formation of collagen for the normal functioning of bones.
  • Vitamin D, Vitamin C, Zinc and Selenium contribute to the normal functioning of the immune system.
  • Table 1 List of the various additional components that the composition object of the invention can comprise, and the quantities in which these components are found in each of the particular and preferred embodiments of the present invention.
  • composition object of the invention are obtained by a method comprising the following steps:
  • composition object of the invention is presented in powder form.
  • composition object of the invention is in powder form
  • composition in accordance with the first aspect of the invention, can be presented in sealed sachets, in themselves conventional in the food and pharmaceutical industry.
  • Another form of presentation of the food supplement, in accordance with the invention is in capsule form, such as conventional gelatin capsules, inside which the composition in powder form is contained.
  • the invention relates to the use of the composition object of the invention as a food supplement for the prevention and delay of aging in animal or human cells.
  • the invention relates to the composition defined herein for use as a medicament.
  • the invention relates to the composition that is defined herein for its use as an anti-aging agent.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective pharmaceutical amount of the composition in accordance with the first aspect of the invention and a pharmaceutically acceptable excipient.
  • an anti-aging agent which can also be called an anti-maturing agent, is a compound that has an ability to fight aging, whose effect it is to delay the effect of aging and the proliferative capacity of cells.
  • the expression "pharmaceutical composition” refers to a formulation that has been adapted to deliver a predetermined dose of one or more useful therapeutic agents to a cell, a group of cells, an organ or a tissue.
  • pharmaceutically acceptable excipient means a therapeutically inactive substance that is said to be used to incorporate the active ingredient and that is acceptable to the patient from a pharmacological/toxicological standpoint and to the pharmaceutical chemist who manufactures it from a physical/chemical standpoint, with respect to composition, formulation, stability, patient acceptance and bioavailability.
  • a food supplement is understood as a food product intended to supplement the normal diet and consisting of concentrated sources of nutrients or other substances that have a nutritional or physiological effect, in single or combined form, is marketed in dosed form, i.e. capsules, pastilles, tablets, pills and other similar forms, sachets of powders, ampoules of liquid, bottles with droppers and other similar forms of liquids and powders to be taken in small unit quantities, as defined in Directive 2002/46/EC of the European Parliament.
  • the use of the composition as a food supplement comprises orally administering an amount between 20mg to 200mg of the composition, 2 to 6 times a day.
  • the use of the composition as an anti-aging agent is indeed the object of the invention, particularly prevention and/or treatment of oxidative stress related with aging
  • the use of the composition as a food supplement comprises orally administering to an individual a dose at least twice a day.
  • this dose should be administered to the patient at least twice a day and a maximum of 6 times a day.
  • the administration of the dose to the patient is 3, 4 or 5 times a day, most preferably 3 times a day.
  • a dose is defined as the amount of medicine containing the exact measure of active ingredient so as to be efficient, effective and safe for the patient, and that solves the health problem for which it has been indicated.
  • a dose has an amount of the composition object of the invention of between 20mg to 200mg.
  • a dose can comprise between 80mg to 150mg of the composition.
  • a dose may comprise 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 20mg, 100mg, 110mg, 120mg, 130mg, 140mg, 150mg, 160mg, 170mg, 180mg, 190mg or 200mg of the composition object of the invention.
  • a dose has an amount of the composition object of the invention of between 1 pg to 20mg. In preferred embodiments, a dose can comprise between 5pg to 20mg of the composition. In particular embodiments of the present invention, a dose may comprise 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 20 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg or 200 pg, 500 pg, 1mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, or 10mg of the composition object of the invention.
  • the composition for use comprises administering to a subject an effective amount of the composition.
  • the amount of compound that prevents and/or treats of oxidative stress related with aging can be an amount from a low of about 0.1 pg/ml, about 0.2 pg/ml, or about 0.5 pg/ml, to a high of about 100 pg/ml, about 200 pg/ml, or about 500 pg/ml.
  • the amount of compound that delays aging can be from about 0.1 pg/ml to about 100 pg/ml, from about 0.2 pg/ml to about 200 pg/ml, from about 0.5 pg/ml to about 500 pg/ml, from about 0.1 pg/ml to about 10 pg/ml, from about 0.1 pg/ml to about 20 pg/ml, from about 0.1 pg/ml to about 50 pg/ml, from about 0.5 pg/ml to about 20 pg/ml, from about 0.5 pg/ml to about 50, from about 0.5 pg/ml to about 100 pg/ml, from about 0.5 to about 200 pg/ml, from about 0.5 pg/ml to about 500 pg/ml, from about 15 pg/ml to about 25 pg/ml, from about 15 pg/ml to about 50, from about
  • preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
  • preventing refers to administering a compound prior to the onset of clinical symptoms of oxidative stress associated with aging so as to prevent a physical manifestation of aberrations associated with oxidative stress related with aging.
  • treatment refers to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • Such measurements and assessments can be made in qualitative and/or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
  • treatment and “treating” refer to the medical management of a subject with the intent to cure, ameliorate, or stabilize oxidative stress related with aging.
  • treatment and “treating” refer to the medical management of a subject with the intent to prevent oxidative stress related with aging.
  • this composition can be administered to the patient dissolved or suspended in a liquid, preferably in an aqueous liquid, and most preferably in beverages such as fruit juices, milk, water.
  • a liquid preferably in an aqueous liquid
  • beverages such as fruit juices, milk, water.
  • it can also be mixed with foods such as yogurt, liquid yogurt, soups, purees, creams, or porridge. These foodstuffs have to be at optimum temperature to be consumed and must never be heated after having added the composition object of the invention.
  • the composition may comprise an abundance relative to all microbial proteins of at least 85% of proteins of Saccharomyces culture lysates. Equally, the composition may comprise a relative abundance of at least 5-0.02% of proteins from culture lysates of each of the following microorganisms: Bacillus, Lactobacillus, Streptococcus, Saccharomyces, and Bifidobacterium.
  • the composition may comprise an abundance relative to the totality of microbial proteins between 1.50 to 9% of proteins from bacterial lysates of the genus Bacillus in percentage by weight, preferably between 2% to 7%, and even more preferably between 4.45% to 6.35%.
  • the relative abundance of proteins from bacterial lysates of the genus Bacillus can be 2%, 2.50%, 3%, 3.50%, 4%, 4.50%, 5%, 5.50%, 6%, 6.50%, 7%, 7.50%, 8%, 8.5% or 9%.
  • the proteins that have been identified from bacterial lysates of the genus Bacillus are selected from the group consisting of Q65HF3, P54944, Q5WEC7, and combinations thereof.
  • the composition may comprise an abundance relative to the totality of microbial proteins between 0.01% to 1.20% of proteins from bacterial lysates of the genus Bifidobacterium in percentage by weight, and preferably between 0.07% to 1.10%.
  • the relative abundance of proteins from bacterial lysates of the genus Bifidobacterium can be 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 0.95%, 1.10%, 1.15% or 1.20%.
  • the proteins that have been identified from bacterial lysates of the genus Bifidobacterium are selected from the group consisting of B8DTX9, B8DSQ4, and combinations thereof.
  • the composition may comprise an abundance relative to the totality of microbial proteins of at least 2% to 8% of proteins from bacterial lysates of the genus Lactobacillus in an amount in percentage by weight preferably between 3.40% to 7.55%, and even more preferably between 4% to 5.45%.
  • the relative abundance of proteins from bacterial lysates of the genus Lactobacillus can be 2%, 2.50%, 3%, 3.50%, 4%, 4.50%, 5%, 5.50%, 6%, 6.50%, 7%, 7.50% or 8%.
  • the proteins that have been identified from bacterial lysates of the genus Lactobacillus are selected from the group consisting of P35829, A0A0H0YNJ3, A5VJ92, Q1G910, and combinations thereof.
  • the composition may comprise an abundance relative to the totality of microbial proteins between 49% to 99.00% of proteins from bacterial lysates of the genus Saccharomyces in an amount in percentage by weight preferably between 65% to 90%, and even more preferably between 75% to 85%.
  • the relative abundance of proteins from bacterial lysates of the genus Saccharomyces can be 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%.
  • the relative abundance of proteins from bacterial lysates of the genus Saccharomyces can be 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • the proteins that have been identified from bacterial lysates of the genus Saccharomyces are selected from the group consisting of E7NI79, E7LV64, AOAOL8VTA7, E7Q2B5, E7NEQ3, A0A0L8VV91 , A0A0L8VIG1 , A0A0L8VIS1 , and combinations thereof.
  • the composition may comprise an abundance relative to the totality of microbial proteins between 0.15% to 0.90% of proteins from bacterial lysates of the genus Streptococcus in percentage by weight, preferably between 0.17% to 89%, and even more preferably between 0.33% to 66%.
  • the relative abundance of proteins from bacterial lysates of the genus Streptococcus can be 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 070%, 0.75%, 0.80%, 0.85% or 0.90%.
  • the proteins that have been identified from bacterial lysates of the genus Streptococcus are selected from the group consisting of Q5M5J3, Q5M2M7, and combinations thereof.
  • the composition object of the invention is obtained from cultures of probiotic microorganism of the microorganisms of the genera described herein, which comprise a certain amount of Colony Forming Units (in English: CFU).
  • a Colony Forming Unit is a term of microbiology. It is an indicator of the amount of live microorganisms present in a medium.
  • one starts from a number of bacteria of the genus Lactobacillus comprising between 28.64% to 66.82% of CFU with respect to the total CFU of the composition, most preferably between 40.66% to 57.36%.
  • probiotic bacteria are cultured under standard conditions, as set out in the culture protocols published by Colection Espanola de Cultivos Tipo (CECT), indicated for each of the bacterial species described in this document.
  • CECT Colection Espanola de Cultivos Tipo
  • lysates consists in combining a non-mechanical method with a mechanical one.
  • the microbial cells undergo heat treatment.
  • Each batch of viable cell culture undergoes a sterilization cycle in an autoclave at 121 °C for 20 to 30 minutes. This temperature denatures and coagulates the proteins by inactivating them. It also causes membrane damage, ribosome aggregation, DNA strand breakdown and enzyme inactivation.
  • composition in accordance with the invention, is the result of the combination of probiotic bacteria that after a process of growth and lysis generates an extract consisting of a set of metabolites, proteins, DNA fragments and other components, such as, for example, peptidoglycans, which dosed efficiently is able to alter and modify in a striking way the host microbiota through several mechanisms, so as to confer an activation of the immune system reversing dysbiosis and strikingly enhancing the delay of cellular aging.
  • the invention relates to the use of a composition of oral administration to prevent and delay aging, that comprises lysates of probiotic microorganisms in the form of dry powder, in the following amount in percentage by weight with respect to the total:
  • the use of the composition is to prevent and delay aging in animal or human cells.
  • the composition comprises the lysates of probiotic microorganisms in the form of dry powder, in the amount as a percentage by weight of the total as disclosed in compositions “A”, “B”, “C”, “D” E”, “F”, “G”, “H” or “I”.
  • the use comprises administering the composition in an amount between 20mg to 200mg of the composition and 2 to 6 times a day orally.
  • the use comprises administering to a subject an effective amount of the composition.
  • the amount of compound that delays aging can be an amount from a low of about 0.1 pg/ml, about 0.2 pg/ml, or about 0.5 pg/ml, to a high of about 100 pg/ml, about 200 pg/ml, or about 500 pg/ml.
  • the amount of compound that delays aging can be from about 0.1 pg/ml to about 100 pg/ml, from about 0.2 pg/ml to about 200 pg/ml, from about 0.5 pg/ml to about 500 pg/ml, from about 0.1 pg/ml to about 10 pg/ml, from about 0.1 pg/ml to about 20 pg/ml, from about 0.1 pg/ml to about 50 pg/ml, from about 0.5 pg/ml to about 20 pg/ml, from about 0.5 pg/ml to about 50, from about 0.5 pg/ml to about 100 pg/ml, from about 0.5 to about 200 pg/ml, from about 0.5 pg/ml to about 500 pg/ml, from about 15 pg/ml to about 25 pg/ml, from about 15 pg/ml to about 50, from about
  • composition object of the invention acts directly on the microbiota in humans. Thanks to this restorative and modulating action, the inventors have confirmed that, strikingly, intervening in the microbiota of human beings can significantly improve the state of health of people, and in particular, slow down the aging process, which materializes in delay and prevention of the development of decreased energy, the change in appearance in humans and animals due to such aging, as demonstrated by the experimental results described herein.
  • the inventors have been able to verify that the oral administration of the composition object of the invention exerts an effect on the adjustment and reprogramming of the microbiota present in the intestinal tract of people, and that it is not just a local effect since the whole organism benefits from this modulation of the microbiota, thus becoming a systemic effect.
  • composition of oral administration to prevent and delay aging characterized in comprising lysates of probiotic microorganisms in the form of dry powder, in the following amount as a percentage by weight of the total: - 5% to 30% of bacterial lysates of the genus Bacillus',
  • composition in accordance with clause 1 wherein the bacterial lysates of the genus Bacillus are of the species that are selected from the group consisting of Bacillus clausii, Bacillus coagulans, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bacillus paralicheniformis, and combinations thereof.
  • composition in accordance with clauses 1 or 2, wherein the bacterial lysates of the genus Bifidobacterium are of the species that are selected from the group consisting of Bifidobacterium animalis subsp lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, and combinations thereof.
  • composition in accordance with any of clauses 1 to 3, wherein the bacterial lysates of the genus Lactobacillus are of the species that are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus Lactobacillus salivarius, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus kefiri, and combinations thereof.
  • composition in accordance with any of clauses 1 to 6, comprising at least one additional component that is selected from the group consisting of Riboflavin, Vitamin C, Manganese, Selenium, Zinc, Vitamin D and Magnesium.
  • composition in accordance with any of clauses 1 to 8 as a food supplement for the prevention and delay of aging in animal or human cells.
  • composition in accordance with clause 9, wherein the composition is administered an amount between 20mg to 200mg of the composition and 2 to 6 times a day orally.
  • composition in accordance with clauses 9 or 10, wherein the composition is presented in the form of dry powder inside watertight sachets or encapsulated in gelatin capsules.
  • composition in accordance with any of clauses 1 to 8, for use as a medicament for use as a medicament.
  • composition in accordance with claim 13 for use in the prevention and delay of aging in animal or human cells.
  • composition in accordance with clauses 12 or 13, wherein the composition is administered an amount between 20mg to 200mg of the composition and 2 to 6 times a day orally.
  • a pharmaceutical composition comprising an effective pharmaceutical amount of the composition in accordance with any of claims 1 to 8, and a pharmaceutically acceptable excipient.
  • composition object of the invention is the following:
  • the present composition includes lysates of microorganisms, that is, it does not present living organisms such that this composition is presented as a safe alternative, since it would avoid the possible dangers of colonizing organisms, and also does not present the toxicity that a conventional drug can present.
  • the process consists in obtaining the mixture of dry microbial lysates of different strains of Bacillus sp., Lactobacillus sp., Bifidobacterium sp., Streptococcus sp. and Saccharomyces sp. To achieve this, these are grown by fermentation, concentrated, broken-up, and finally the cellular components are dried.
  • the postbiotic formulation will be the mixture of lysates that can be accompanied by food ingredients in a capsule or powder supplement format.
  • an inoculum is prepared in a culture medium in a flask of each microorganism, in adequate quantity and volume to be able to proceed to inoculate the fermenter with respective incubated inoculums.
  • the microorganisms preserved in vials with 20% glycerol at -80°C are immersed in a water bath at 30°C to allow a quick and complete thawing of the respective microorganisms.
  • the fermenters are sown directly separately.
  • the percentage of sowing is 3% for Bacillus species and 5% for each species of Bifidobacterium, Lactobacillus, Streptococcus and Saccharomyces.
  • the inoculum is grown in Nutrient Broth (NB) at 30°C for 15h at 250 rpm. It is cultured in a fermentation medium composed of Glucose H 2 O 25g/l, Yeast extract 5 g/l, K2HPO4 2.5 g / 1, MgSOr FW 5g/l, Tween 80 1g/l, at 30°C ⁇ 1°C throughout the process and pH 6.8 ⁇ 0.1 by sterile addition of NH4OH 25% or H3PO4 35% automatically. Dissolved oxygen is maintained >40% by stirring (150-250 rpm) and aeration (1-1.5vvm) with a working pressure of 0.5 atm. After approximately 20-24h, the base consumption (NH4OH) is stopped, and the temperature is lowered to 4-8°C to stop cell metabolism and harvest the broth.
  • NH4OH base consumption
  • the inoculum is grown in Tryptic Soy Broth (TSB) at 37°C with a stirring of 150 rpm for 24h.
  • the fermentation medium consists of Glucose 25g/l, Milk powder 8g/l, Yeast extract 5g/l, K2HPO4 2 g/l, KH2PO4 2g /I, MgSC W 0.5g/l, (NH 4 )SO 4 0.5g/l.
  • the growing temperature is 37°C ⁇ 1°C and the pH is maintained at 6.8 ⁇ 0.1 throughout the process by sterile addition of NH4OH 25% or H3PO4 35% automatically, stirring is 150 rpm.
  • the temperature is lowered to 4-8°C to stop the cellular metabolism and harvest the broth.
  • the growth of the inoculum of Bifidobacterium and Lactobacillus is done in De Man, Rogosa, Sharpe Broth (MRS Broth) at 37°C for 24h.
  • the fermentation is carried out at 37°C ⁇ 1°C, Ph 6.2 ⁇ 0.1 and 6.4 ⁇ 0.1 , respectively, with the sterile addition of NH 4 OH 25% or H3PO4 35% automatically, and 50 rpm (the minimum value that guarantees the correct homogenization of the components of the medium).
  • the culture medium is composed of Glucose H2O 20g/l, Yeast extract 5g/l, K2HPO42g/l, Casein peptone 10g/I, Milk powder 8g/l, Sodium acetate 5g/l, Diammonium citrate 2g/l, Mn SO4- H2O 0.05g/l, MgSO4 7H2O 0.2 g/l, Tween 80 1g/l.
  • nitrogen is bubbled until the concentration of dissolved oxygen is at 0%, repeating the operation whenever necessary to maintain the O2 at that level throughout the process.
  • it is lowered to 4-8°C to stop cell metabolism and harvest the broth.
  • the inoculum of S. cerevisiae is grown in YPD for 24h.
  • the temperature is maintained at 30 °C and Ph 5 at 150 rpm and 1.5vvm.
  • the fermentation is stopped in the fermenter itself, lowering the temperature to a value between 4-8 °C.
  • the recovery of the biomass obtained based on the fermentation of each of the microorganisms is carried out by centrifugation or microfiltration, obtaining wet recovered biomasses, concentrating them as many times as necessary to reach between 1.00E+09 to 1.00E+11 CFU/ml in the concentrated biomasses of each microorganism.
  • the rupture or lysis of the cells of each of the wet recovered biomasses is carried out with a heat treatment and sonication, obtaining respective bacterial lysates that are subsequently dried separately in a lyophilizer or atomizer.
  • the dry bacterial lysates are mixed in a mixer, to obtain the modulating composition.
  • Each batch of viable cell culture undergoes an autoclave sterilization cycle at 121°C for 20 minutes. Subsequently, they are subjected to sonication for 20min, at an output power of 500 W with an amplitude of 40% and 10 seconds of pause obtaining a mass of lysed cells of the probiotic (Qsonica, Q500).
  • the final solution is freeze-dried and ground to obtain a lysate of the powdery probiotic microorganism.
  • the powder is kept in a cool environment away from heat.
  • lysates 0.5 gr of powder stock is taken from each batch and dissolved in 10 ml of distilled water. From this suspension a 1 :8 dilution is prepared (100 ul sample and 700 ul water) and centrifuged for 5 minutes at 5000 rpm. The supernatant is collected, and 1.5 ul of sample is placed in a cuvette to measure absorbance at 260nm, 230nm and 280nm in a Nanodrop Tecan Spark 10M, using distilled water as a target. The measure is at least duplicated and usually repeated more than four times.
  • the amount of DNA present in each of the lysates is as follows:
  • composition object of the invention was prepared with the following dry bacterial lysates in the following proportions:
  • Example 2 MTT assay and determination of telomerase activity by QTRAP
  • toxicity was determined by the MTT assay and the evaluation of the effect of a probiotic extract of Igen Biolab, the composition of which is indicated in Table 4, on telomerase activity by Q-TRAP in cultures of primary human fibroblasts.
  • Table 4 Composition of 1 sample of the composition object of the invention. The percentage by weight of each of the bacterial species comprising the dry bacterial lysates described in this document is shown. The table also indicates the number of colony forming units.
  • the MTT toxicity assay is a colorimetric test to measure the metabolic activity of cells by serving as a substrate for cellular enzymes that reduce the orange tetrazolium dye, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to its insoluble formazan, giving a purple color.
  • the conversion rate is directly proportional to the mitochondrial metabolic activity of cells via NAD (P) H-dependent cellular oxidoreductase enzymes and serves as a surrogate marker of cell viability.
  • the stock of primary adult human fibroblast cultures (3 passages) was established under standard culture conditions. The cells were seeded at 3x10 3 cells/ cm2 in a fibroblast medium (Innoprot kit).
  • Fibroblast Medium is a complete medium designed for the optimal growth of human fibroblasts in vitro. It is a sterile liquid medium containing essential and non- essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and a low concentration of fetal bovine serum (2%).
  • the medium is HEPES and bicarbonate buffer system with a pH of 7.4 after being balanced in an incubator with an atmosphere of 5% CC>2/95%.
  • the medium is formulated (quantitatively and qualitatively) to provide a defined and optimally balanced nutritional environment that selectively promotes the proliferation and growth of normal human fibroblasts in vitro.
  • Previously expanded cells were seeded into 96-well plates (Nunc) at 0.5x10 4 cells/plate and 0.35x10 4 cells/plate during a 72-hour and one-week treatment, respectively. This concentration shows the best window for the MTT assay, with better sensitivity and low variability.
  • the compound was supplied in dry powder form and remained in optimal condition until use. An amount of 18 mg of the compound (powder) was dissolved in 18 ml (1 mg/ml) of 20 % DMSO at room temperature. Eight-point curves were prepared, dilutions 1/2 (highest concentration 1 mg/ml). The final DMSO concentration was 0.5% (this concentration does not affect cells).
  • the cells were washed once with PBS and treated with the compounds in their respective cell culture media (fibroblast media kit). Each condition was analyzed in triplicate with and without H2O2 10 pM. For positive and negative controls, methyl methane sulfonate (MMS) 8mM and 100% DMSO were used, respectively.
  • MMS methyl methane sulfonate
  • the plates were incubated for 72 hours and a week, while the medium with the compound was changed every other day. After the treatment period, the cells were washed twice with PBS and the medium was replaced with MTT reagent at 0.5 mg/ml in DMEM without phenol red. The plates were gently shaken and incubated for 4 hours. After incubation, the medium was removed and replaced by DMSO. The plates were gently shaken to solubilize the formazan crystals.
  • Toxicity Test in fibroblasts at 72 hours and one week.
  • MTT assay results expressed as the percentage of cell death after treatment, after 72 hours under standard conditions. after 72 hours on oxidative stress. after one week under standard conditions. after one week on oxidative stress.
  • the concentrations of the composition of the invention chosen for the Q-TRAP and TAT assays were 50, 16.7 and 5.6 pg/ml.
  • This assay determines telomerase activity by Q-TRAP in adult human fibroblast primary cultures after 6, 24, 48 and 72 hours of treatment at the above concentrations.
  • Q-TRAP can measure relative telomerase activity using the Telomere Repeated Amplification Protocol (TRAP), modified for quantitative real-time PCR analysis (Q- TRAP). This method has the advantages of greater sensitivity, speed and a high- performance format compared to the normal TRAP assay. The activity of the enzyme telomerase in lysates of whole cells of cell cultures is evaluated.
  • TRAP Telomere Repeated Amplification Protocol
  • Q- TRAP quantitative real-time PCR analysis
  • the general mechanism of the Q-TRAP technique consists of the lysis of cell granules for the extraction of proteins, which are then quantified and stored under specific conditions to prevent their degradation.
  • the protein obtained in the process was used within 24 hours, the samples were stored at 4°C.
  • Telomerase protein extracts are incubated with a specific oligonucleotide substrate to allow enzymatic addition of telomeric DNA repetitions by endogenous telomerase.
  • telomerase extension products are amplified and quantified by real-time qPCR.
  • Ct cycle threshold
  • a positive reaction is detected by the accumulation of the fluorescent signal.
  • the Ct cycle threshold
  • the telomerase-positive standard dilution series is plotted against telomere protein concentration (r2 > 0.9) as a standard curve of Ct values.
  • test is performed in triplicate.
  • the mean and standard deviation (SD) of each triplicate are calculated, which include both positive (standard lymphoid cell line curve) and negative (heat-inactivated) controls.
  • Protein concentration protein quantification is performed on each sample using the Biorad protein assay. A minimum protein concentration of 0.3 pg/pl is required to proceed with sample analysis to ensure consistent results.
  • Regression curve internal controls are included, and a regression analysis is performed for each cycle/plate. It is repeated in the plates in which the regression curves have an R2 less than 0.9
  • T able 10 Protein concentration results for samples analyzed with Q-TRAP. Each column shows the concentration of each triplicate.
  • Standard curve results generated by graphically representing the threshold cycles (Ct values) of the HeLa cell line standards versus the registry.
  • the measurements were made in triplicate to calculate the coefficients of variation and the mean of the amplification signals.
  • Table 12 The T-Student analysis indicates whether there are significant differences between the QTRAP results compared to the control group. Significant differences are indicated in the "Significance" column. Lowest to highest: No: not significant; Yes (*): p ⁇ 0.05; Yes (**): p ⁇ 0.01 ; Yes (***): p ⁇ 0.001 ; Yes (****): p ⁇ 0.0001.
  • Group IG_1 has a higher RTA than the control group for all the times. Although the differences are not statistically significant, the effect maintained on activation for all points shows a consistent positive effect for the I G_1 group.
  • the IG_2 group presents a telomerase activation for the times 12, 24 and 72 hours.
  • the IG_3 group presents higher RTA compared to the untreated control for times 12 and 72.
  • Example 3 Proliferation Analysis in Cell Culture and Telomere Length Measurements by TAT® 3.1. Proliferation analysis in cell culture
  • Fibroblast Medium is a complete medium designed for optimal growth of normal human fibroblasts in vitro. It is a sterile liquid medium containing essential and non-essential amino acids, vitamins, organic and inorganic compounds, hormones, growth factors, trace minerals and a low concentration of fetal bovine serum (2%).
  • the medium contains HEPES and buffered bicarbonate and has a pH of 7.4 when balanced in an incubator with an atmosphere of 5% CC>2/95% of air.
  • the medium is formulated (quantitatively and qualitatively) to provide a defined and balanced nutritional environment that selectively promotes the proliferation and growth of normal human fibroblasts in vitro.
  • the media are renewed every 2-3 days and the cells are passed in subconfluence (70-80%) every 7 days.
  • Compounds or treatment are added to cells in culture.
  • Cell growth is monitored for each condition by counting the number of cells at each step using a CountessTM (Invitrogen) cell counter.
  • PD Population doubling
  • a PD is equivalent to one round of cell replication.
  • telomere length is measured using a high- throughput (HT) Q-FISH technique.
  • HT high- throughput
  • telomeres are hybridized with a fluorescent peptide nucleic acid (PNA) probe that recognizes three telomere repetitions (sequence: Alexa488-OO- CCCTAACCCTAACCCTAAA, Panagene). Images of nuclei and telomeres are captured by a high-content display system (see below).
  • the fluorescent signal strength of telomeric PNA probes that hybridize with each telomere is proportional to the length of that telomere. Fluorescence intensities are translated into base pairs through a standard regression curve that is generated using control cell lines with known telomere length.
  • Sample preparation and HT Q-FISH On the day of processing, samples and control cell lines frozen in liquid nitrogen are thawed at 37°C and cell counts and viability are determined. Aliquots with a viability of less than 60% are considered below our quality control standards and will not be further analyzed.
  • Cells are seeded into 384-well plates at a density of 15,000 cells per well with 5 replicates of each sample and 8 replicates of each control cell line. Two identical independent plates are prepared for each set of samples. The cells are fixed with methanol/acetic acid (3/1 , vol/vol). Once the cells have fixed themselves in the plate, they are treated with pepsin to digest the cytoplasm and the nuclei are processed for in-situ hybridization with the PNA probe. After several washing steps and incubation with standard DAPI for DNA staining, the wells are filled with mounting medium and the plate is stored overnight at 4°C.
  • HT Microscopy Quantitative image acquisition and analysis is performed on an Opera Phenix high-content screening system (Perkin Elmer), using Columbus software, version 2.9 (Perkin Elmer). Images are captured using a 40 x 0.95 NA water immersion lens. UV wavelengths excitation and 488 nm are used to detect the DAPI and A488 signals respectively. With constant exposure settings, 15 independent images are captured in different positions for each well. The images of the nuclei are then used to define the region of interest of each cell, measuring the fluorescence intensity of the telomeres of the A488 image in all of them. The intensity results for each focus are exported to the Columbus 2.4 (Perkin Elmer) software. Telomere length distribution and median telomere length are calculated with Life Length's proprietary software. The statistical analysis of the data was performed using the T-student test.
  • TAT fluorescence intensity values to telomere length measurements is achieved by performing TRF (terminal restriction fragmentation) on six human lymphocyte cell lines (calibration/method comparison). The same set of samples is analyzed by both TAT and the TRF (Definition of Systemic Error TAT) reference method.
  • VALIDATION DATA shows a correlation of 0.99.
  • VALIDATION DATA indicates that TAT has a standard deviation of 454 base pairs.
  • VALIDATION DATA defines the detection limit at 800 base pairs and demonstrates very high specificity.
  • Reportable range of the median an analysis of the median telomere length of 6 cell lines is performed that covers the reportable range and defines its lower and upper limits.
  • VALIDATION DATA sets the lower level at 4,700 base pairs and the upper level at 14,400 base pairs.
  • Reference range The analysis of median telomere length has been evaluated in hundreds of human samples to define the reference range of TAT and its percentiles (5th, 10th, 25th, 50th, 75th and 95th) for the different ages.
  • the VALIDATION DATA established population curves - normal population curve from 18 to 85 years, to extrapolate and generate reports.
  • regression curve Internal controls are included, and a regression analysis is performed for each sample/plate. It is repeated in the plates where the regression curve has an R2 less than 0.92.
  • the number of spots analyzed per sample must be greater than 10,000.
  • telomere Length results by TAT The table below shows the median telomere length and median telomere length of the 20th percentile (both in base pairs, bp) for each sample, as well as the percentage of short telomeres. The latter is defined as the percentage of telomeres with a length less than 3 Kbp ( ⁇ 3 Kbp). All measurements were performed fivefold. conditions 3.2.4 Analysis of results
  • telomere shortening was one of the main causes of telomere shortening.
  • T-Student analysis indicates if there are significant differences in the telomere shortening ratio by observing each treatment and time. Significant differences are indicated in the "Significance" column. From lowest to highest significance: No: no significance; Si (*): p ⁇ 0.05; Si (**): p ⁇ 0.01; Si (***): p ⁇ 0.001; Yes (****): p ⁇ 0.0001
  • T-Student analysis indicates if there are significant differences in the telomere shortening ratio by observing each treatment and time. Significant differences are indicated in the "Significance" column. From lowest to highest significance: No: no significance; Si (*): p ⁇ 0.05; Si (**): p ⁇ 0.01 ; Si (***): p ⁇ 0.001 ; Si (****): p ⁇ 0.0001.
  • IG_2 and IG_3 show a reduction in the telomere shortening ratio when compared to the control group. Although this effect is not apparent at week 4, the positive effect can be observed again at week 6 (for IG_2) and week 8 (for IG_2 and IG_3) suggesting a protective effect on telomeres. The reduction in shortening at week 8 was statistically significant. • Oxidative Stress Conditions
  • the IG_1 , IG_2 and IG_3 groups showed an improvement in reducing the telomere shortening rate.
  • the IG_1 and IG_3 groups continue to maintain this effect, where the IG_1 group also presents statistically significant differences.
  • Example 2 A sample of the composition was evaluated in accordance with the invention corresponding to the composition analyzed in Example 2 (hereinafter also referred to as “Sample”).
  • a stock solution was prepared with distilled water, performing serial dilutions to obtain, finally, different final concentrations in the plate of NGM medium (Nematode Growth Medium), used for the cultivation of C. elegans.
  • NGM medium Nematode Growth Medium
  • the product was added on the surface of the agar.
  • the trials were conducted with the wild strain of C. elegans (N2). Populations synchronized in age were cultured, collecting the embryos in the different culture dishes:
  • the plates were incubated at 20°C and nematode survival counts were performed in each condition, with periodic transfers from the study population every two days to a new medium. During this period, a viability count was carried out, to finally obtain survival curves in each condition.
  • worm samples 25/condition and assay
  • locomotion was determined by quantifying the number of sinusoidal curvatures over 40 seconds.
  • a total of 15 worms/condition were analyzed, depositing at the same time 5 worms in the center of the NGM plate and determining their position after 2 minutes (zone 1 closest to the center, 2 or 3 farther from the center).
  • the trials were conducted with the wild strain of C. elegans (N2). Age-synchronized worms were obtained from the incubation of gravid adults in the corresponding culture plates. The embryos were incubated at 20°C under different culture conditions, NGM (control) and NGM supplemented with the sample at different doses:
  • Sample medium (0.05; 0.1 ; 0.5; 1 , 5, 10, 25, 50, 100, 200 and 400 pg/mL).
  • the worms were incubated in the NGB medium (control) in the NGM + Vitamin C medium, as well as under the different conditions in the NGB + Sample medium at 20°C. Subsequently, 5-day-old adults were transferred to culture medium plates with hydrogen peroxide and incubated at 20°C. Finally, the survival of the worms in each condition was determined.
  • Figure 13 shows the effect of the high-dose Sample on the longevity of C. elegans N2.
  • the value of average life expectancy (Time in which 50% of the population is alive) for the control condition (NGM) and the Sample at 1 mg/mL are indicated with dashed lines.
  • Figure 14 shows the survival curves of C. elegans (N2) obtained in populations treated with the Sample (1 mg I mL) using the GraphPad Prism software.
  • Table 24 shows the statistical data of the survival curves of C. elegans after treatment with the Sample at the dose of 1 mg/mL
  • Figure 15 represents the average sine waves for each of the different treatment doses.
  • the treatment did not improve the mobility of C. elegans, as can be seen in Figure 15, showing the average mobility of C. elegans populations with the Sample at different doses.
  • the data are for a single trial.
  • Figure 18 showing the degree of dispersion of worms treated with the Sample represents the data obtained with adult worms treated with the product doses of 0.1 ; 0.5 and 1 mg/mL. It was determined that the dose of 1 mg/mL caused a greater accumulation of worms in zone 3 (most distal of origin), indicating a greater degree of dispersion of worms in this condition. In addition, it was observed that as we increase the concentration of the product, a greater number of worms accumulate in zone 3.
  • Table 25 shows the percentage increase in survival and P-value obtained (ANOVA test) of each of the conditions of the Sample with respect to the NG control condition.
  • Table 25 percentage increase in survival and P-value obtained (ANOVA test) of each of the conditions of the Sample with respect to the NG control condition.
  • the most effective dose was 0.5 pg/mL (23% increase in survival vs. control condition; P-value ⁇ 0.001) (Table 23).
  • the dose of 0.1 pg/mL caused a 9% increase in survival vs control (P ⁇ 0.01) (Table 23).
  • Figure 21 represents the relative percentage increase in survival of C. elegans with the effective dose (0.5pg/mL) of the Sample versus control condition, indicating an increase of 88.5%.
  • the example shows a summary of the functional activity in C. elegans of the analyzed Sample. It can be seen that the Sample has been functionally characterized in the preclinical model of C. elegans, showing some effect on longevity (at high doses), a very significant effect on mobility and a high antioxidant activity (at very low doses).
  • Table 26 Composition of sample 2 of the composition object of the invention.
  • the percentage by weight of each of the bacterial species comprising the dry bacterial lysates described in this document is shown.
  • the oxidative stress activity and antiaging activity were evaluated in the same conditions as with Sample 1 in Example 3 and 4 and the results obtained were very similar.

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Abstract

L'invention concerne une composition d'administration orale destinée à être utilisée dans la prévention et/ou le traitement du stress oxydatif associé au vieillissement, comprenant des lysats de micro-organismes probiotiques sous la forme de poudre sèche du genre Bacillus ; Bifidobacterium ; Lactobacillus ; Saccharomyces et Streptococcus.
PCT/EP2023/078516 2022-10-14 2023-10-13 Composition d'administration orale obtenue à partir de lysats de micro-organismes probiotiques destinés à être utilisés en tant qu'agent anti-vieillissement WO2024079325A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003155234A (ja) 2001-11-20 2003-05-27 Fancl Corp 抗老化組成物
US8492353B2 (en) 2002-08-06 2013-07-23 Otsuka Pharmaceutical Co., Ltd. Antiaging composition
ES2783723A1 (es) * 2019-03-15 2020-09-17 Igen Biolab Group Ag Composicion obtenida de lisados bacterianos para modular el microbioma humano, formulacion terapeutica y suplemento alimenticio que comprenden la composicion, y usos de la composicion.
US20200345694A1 (en) 2017-10-27 2020-11-05 Amorepacific Corporation Antiaging composition comprising cytochalasin d or sag, and method for screening antiaging substance

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003155234A (ja) 2001-11-20 2003-05-27 Fancl Corp 抗老化組成物
US8492353B2 (en) 2002-08-06 2013-07-23 Otsuka Pharmaceutical Co., Ltd. Antiaging composition
US20200345694A1 (en) 2017-10-27 2020-11-05 Amorepacific Corporation Antiaging composition comprising cytochalasin d or sag, and method for screening antiaging substance
ES2783723A1 (es) * 2019-03-15 2020-09-17 Igen Biolab Group Ag Composicion obtenida de lisados bacterianos para modular el microbioma humano, formulacion terapeutica y suplemento alimenticio que comprenden la composicion, y usos de la composicion.
ES2783885A1 (es) * 2019-03-15 2020-09-18 Igen Biolab Group Ag Procedimiento de obtencion de una composicion moduladora del microbioma intestinal humano

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KUMAR RAVI ET AL: "Cell-Free Culture Supernatant of Probiotic Lactobacillus fermentum Protects Against H2O2-Induced Premature Senescence by Suppressing ROS-Akt-mTOR Axis in Murine Preadipocytes", PROBIOTICS AND ANTIMICROBIAL PROTEINS, vol. 12, no. 2, 22 July 2019 (2019-07-22), New York, NY ; Heidelberg : Springer, pages 563 - 576, XP093135351, ISSN: 1867-1306, Retrieved from the Internet <URL:http://link.springer.com/article/10.1007/s12602-019-09576-z/fulltext.html> [retrieved on 20240227], DOI: 10.1007/s12602-019-09576-z *
SHARMA ROHIT ET AL: "Probiotic bacteria as modulators of cellular senescence: emerging concepts and opportunities", GUT MICROBES, vol. 11, no. 3, 10 December 2019 (2019-12-10), United States, pages 335 - 349, XP093103697, ISSN: 1949-0976, DOI: 10.1080/19490976.2019.1697148 *

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